Field of the Invention
The present invention relates to a focusing method, a measuring method, a principal point detecting method, a focusing device, a measuring device, and a principal point detecting device.
Description of the Related Art
A contrast-detect autofocus method is known as a method of focusing on a sample at the time of observing the sample with a microscope. The contrast-detect autofocus method is a focusing method suitable when a sample has contrast. Japanese Patent Application Laid-open No. S64-54408 describes a contrast-detect autofocus method in the section of Related Art.
In this focusing method, the image contrast at the present position is compared with the contrast at a position that the lens is moved to for a predetermined time, the distance to move next time is determined from the slope (contrast change/moving distance) at this point of time, and when the distance to move becomes a certain value or smaller due to the reduction in the contrast change, the position of the lens is set as a focusing position.
Samples observed with a microscope are, for example, cells. Although cells are colorless and transparent, contrast is imparted to cells by staining. Therefore, as for stained cells, it is possible to focus on the cells by using the contrast-detect autofocus method. By contrast, when cells in a living state are to be observed, it is not preferable to stain the cells. In this case, cells that are alive (hereinafter referred to as “living cell” as appropriate) are colorless and transparent and therefore have no contrast. Accordingly, as for living cells, it is difficult to focus on living cells even using the contrast-detect autofocus method.
A phase-contrast observation method is known as a method of observing a colorless and transparent sample. Some of living cells have protrusions and depressions on the surfaces, like phase-type diffraction gratings. Since the phase is changing in such a living cell, non-diffracted light and diffracted light are produced from the living cell when the living cell is illuminated with light. In the phase-contrast observation method, the change in phase is converted into a change in brightness using the interference between non-diffracted light and diffracted light. Specifically, the phase of non-diffracted light is matched with the phase of diffracted light using a phase plate.
In the phase-contrast observation method, even when the sample is colorless and transparent, a sample image with enhanced contrast can be obtained. Then, even when the sample is colorless and transparent, it is possible to focus on the sample by combining the phase-contrast observation method with the contrast-detect autofocus method.
In the phase-contrast observation method, however, a bright rim called halo may appear around the periphery of the image. The brightness of this halo may be extremely high even in a state in which the sample is out of focus. Thus, when a halo occurs, it is difficult to focus on the sample in the contrast-detect autofocus method.
With a method different from the phase-contrast observation method, it is possible to match the phase of non-diffracted light with the phase of diffracted light. In another method, the position of the sample is displaced from the focus position of the objective lens by a predetermined distance. In this case, contrast Coff when the sample is displaced from the focus position of the objective lens is compared with contrast Con when the sample coincides with the focus position of the objective lens, and then Con<Coff holds. Japanese Patent Application Laid-open No. 2008-20498 discloses a focusing method using such a difference in contrast.
In the focusing method in Japanese Patent Application Laid-open No. 2008-20498, while the sample and the focus position of the objective lens are relatively moved, the following (I) to (III) are performed: (I) acquisition of a differential image using an image before movement and an image after movement; (II) calculation of the contrast value of the differential image; and (III) determination as to whether the contrast value of the differential image is maximum. It is then determined that the sample is in focus when the contrast value of the differential image is maximum.
The focusing method in Japanese Patent Application Laid-open No. 2008-20498 uses the interference between non-diffracted light and diffracted light, similarly to the phase-contrast observation method. In the focusing method in Japanese Patent Application Laid-open No. 2008-20498, however, a halo does not occur as in the phase-contrast observation method. Therefore, it is possible to expand the target range that can be focused.